1. Field of the Invention
The invention generally concerns reagents and methods useful for membrane receptor-ligand binding assays.
2. Description of the Related Art
Much of modern pharmacology and biochemistry is focused on the interactions between different types of biological signaling molecules and their corresponding membrane receptors. Structurally, these membrane receptors are often G-Protein Coupled Receptors (GPCR) from the 7-transmembrane (7tm) protein family, and other types of related 7-transmembrane proteins such as ion channels.
Biological signaling molecules and membrane receptors are present in many physiological processes, and are particularly important for nervous system function. Indeed, nervous system membrane receptor systems, such as the dopamine, serotonin, and opioid receptor families, have been found to be involved in many mental disorders, such as anxiety, depression, and drug abuse. Furthermore, these membrane receptors have been found to be excellent drug targets. GPCR reactive drugs are involved in many other biological processes as well. Kenakin, Annu Rev Pharmacol Toxicol 2002;42:349-79).
To date, however, only a small number of the many possible interactions between the millions of potential candidate drug ligands, and the thousands of different membrane receptors, have been well characterized. With the recent advances in both genomics and nucleic acid microarray technology, the cellular distribution and sequence of these membrane receptors and receptor families are now readily available. As a result, one of the key challenges going forward is to utilize this new knowledge to aid in the development of next generation drugs.
Modern drug discovery and development is a multi-step process. Usually, one or more medically important target receptors are identified, a large number of prototype lead drugs are synthesized, and appropriate High Throughput Screening (HTS) assays are conducted to assess the proper differential binding to an initial set of selected target and non-target receptors. Those lead candidates that survive the process are then subjected to progressively more expensive and stringent assays; whole cell assays, animal studies, Absorption, Distribution, Metabolism, Excretion, Toxicity (ADMET) studies, and finally human Phase I, II, and III studies. The expense and time involved in the later stages, typically hundreds of millions of dollars and many years, are such that it is enormously important that optimal leads be found as early in the process as possible.
One of the best ways to exploit the recent advances in genomics is to use the information to clone and express these membrane receptors in a pure state, and use these cloned receptors for HTS lead identification and optimization. In theory, the later stages of drug development could be significantly streamlined if low-cost and efficient HTS methods were developed to initially optimize a lead""s specific binding to its target receptor, and to detect any unwanted cross-reaction with non-target receptors.
At present, however, membrane HTS screening methods are sub-optimal. Membrane proteins are hydrophobic, and typically only assume their correct physiological conformation in a lipid bilayer membrane environment. Moreover, many membrane receptor proteins rely upon certain aspects of lipid bilayers, such as receptor lateral mobility, association with lipid rafts, association with other proteins, small molecule cofactors etc., for proper function. Such faithful recreations of native membrane structures are difficult to reproduce in a synthetic, in-vitro, environment.
As a result, present membrane HTS screening methods usually rely upon the binding of radioactively labeled ligands to natural membrane receptors (e.g. receptors obtained from natural sources such as cultured cells). These natural membrane receptors are often bound to a solid phase, such as a filter or microwell plate. Radioactive ligand is applied to the sample, followed by one or more washing steps. The bound radioactive ligand is then detected by its radioactive scintillation signal. Thus relatively large quantities of membrane proteins and candidate drug ligands are required for each assay. For example, even the optimized Packard Bioscience FlashPlate(trademark) system requires 25 to 50 ul of fluid for each assay point.
Although the use of natural membranes has strong physiological merit, it is slow, expensive, and cumbersome. An alternate membrane receptor HTS methodology that could return physiologically useful data with cloned membrane receptors would be highly advantageous, as it would enable the many recent genomic insights to be easily and rapidly used.
An ideal membrane-receptor HTS methodology would have a number of other characteristics as well. At present, HTS methods are typically restricted to large, well-financed, commercial organizations. This is because the present methodologies require the use of large quantities of expensive membrane receptors, expensive synthetic drug candidates, and expensive automation. If alternative methods could be devised to reduce the quantities of receptors and synthetic drug candidates by several orders of magnitude, the financial and logistical burden of HTS studies would be greatly eased. This would enable a much larger number of receptors and drug candidates to be screened, and could also make HTS methods feasible in smaller scale settings, such as academic laboratories.
One good way to accomplish this goal is by the development of suitable membrane receptor microarray technology.
Microarray technology: In recent years, microarrays have become widely used for genomic and proteomic biotechnology, biomedical research, and biomedical diagnosis. In particular, microarray methods have become widely used for nucleic acid research, and a large number of nucleic acid microarrays are commercially available from Affymetrix Inc., Incyte Pharmaceuticals, Inc., and many other companies. These methods (reviewed in Schena, Microarray Biochip Technology (2000) Eaton Publishing. Natick, Mass.) generally work by binding a large number of nucleic acid microsamples to the surface of a flat support. Samples containing one or more unknown complementary nucleic acids are then exposed to the nucleic acid microarray, and the sample is allowed to hybridize to the microarray. Hybridized nucleic acids are then detected by various means, and the overall nucleic acid composition of the unknown sample is assessed.
The general principle has been that to detect two biologically interacting elements that form a pair, such as complementary nucleic acid strands, the microarray will contain one-half of the pair, the unknown analyte will contain the other half of the pair, and the interaction between the two elements will generate a detectable signal.
A good overview of protein microarray technology in general, as it exists at the time of this patent application, can be found in the article by Mitchell, xe2x80x9cA perspective on protein microarraysxe2x80x9d, Nature biotechnology (20), 2002, 225-229, the contents of which are incorporated herein by reference.
There have been some previous attempts to produce membrane receptor microarrays, such as U.S. Pat. No. 6,228,326; and Salafsky et. al., Biochemistry (1996) 35: 14773-14781. One approach, pioneered by Boxer and coworkers (Groves et. al., Science (1997), 275: 651-653) relies upon the formation of an artificial planar lipid membrane parallel to a microarray surface. The microarray uses a series of mechanical barriers to separate one lipid region from another, enabling multiple regions to be patterned. The microarray is analyzed by fluorescence recovery after photobleaching (FRAP) techniques. Here, fluorescent membrane components are exposed to a localized region of intense laser irradiation. Following irradiation, a bleached region forms, which can be observed by a fluorescence microscope. This gradually disappears as unbleached membrane components from surrounding regions gradually migrate into the bleached region.
Although useful for demonstration purposes, one fundamental critique of this approach is that due to the close (1 nm) association between the inner leaflet of the artificial membrane lipid bilayer, and the solid phase support, the physiology of the artificial membrane is highly distorted. In particular, larger membrane proteins, such as GPCR proteins, are large enough that they can interact with the solid phase support, resulting in distorted conformations and inactivity. Additionally, the narrow aqueous layer is too thin to enable GPCR proteins to interact with other cytoplasmic side cofactors, which play a key role in the proper function of the receptor.
A second critique of this approach is that FRAP detection techniques are too crude to spot many interesting types of interactions. For example, these methods are of marginal utility for drug discovery purposes. This is because the binding of the relatively small (low molecular weight) drug candidates to relatively large (high molecular weight) membrane receptors will have a negligible impact on the FRAP mobility of the receptors, and thus will be poorly detected by FRAP techniques.
An alternative type of membrane microarray has been proposed by Ziauddin and Sabatini that relies on DNA uptake (Ziauddin J, Sabatini D M, xe2x80x9cMicroarrays of cells expressing defined cDNAsxe2x80x9d, Nature, 2001, 411:107-10). Here, DNA coding for receptor genes is spotted on a microarray surface. After spotting, cells are cultured on the microarray. Those cells located above the DNA spots take up the DNA, and express the appropriate receptor on the cell surface. This is a relatively new approach, and more work will be needed before the relative utility of such microarrays can be accurately assessed.
The xe2x80x9cL1xe2x80x9d chip produced by Biacore Corporation represents a third technique (Williams, C., Cook S., Knoppers M., xe2x80x9cAdvances in Lipid Immobilization: The L1 Chipxe2x80x9d, BIAjournal 2000, 7 (1)). In this technique, membrane fragments from mammalian cells are bound to the surface of a flow-cell. Samples containing potential ligands are injected into the flow cell, and after a washing step, retained material is eluted and analyzed by mass spectroscopy.
Although this technique is clearly useful for certain analytical needs, it has some drawbacks. The present L1 chip design has hydrophobic moieties on the surface of the chip. These interact with membrane samples, causing liposomes and membrane vesicles to rupture. As a result, the applied membranes become firmly attached to the surface of the chip with no aqueous separation layer (Erb E, Chen X, Allen S, Roberts C, Tendler S, Davies M, Forsen S. xe2x80x9cCharacterization of the surfaces generated by liposome binding to the modified dextran matrix of a surface plasmon resonance sensor chipxe2x80x9d Anal Biochem 2000, 280(1):29-35). Thus, as is the case for the Boxer design, ligand-binding reactions are distorted. Additional drawbacks are the relatively slow throughput of the single element flow-cell design, and a large number of false positives produced by the mass-spectrometer detection methodology.
Evanescent wave technology: Evanescent waves are generated when a light wave undergoes total internal reflection at a surface (a junction between two media with different indexes of refraction). A light wave travels through the first media until it encounters the surface boundary, and, at the appropriate angle of incidence (the critical angle), bounces back from the surface and continues traveling through the first media, rather than entering into the second media. Here, a small amount of energy, termed an evanescent wave, penetrates an extremely short distance into the second media, and rapidly decays in intensity as distance from the boundary increases. For optical frequencies and media types commonly used in biological research, the evanescent wave decreases in intensity by about 1/e (that is, about 1/2.71) over a distance of about 260 nm. Thus, over distances of a few hundred nanometers, evanescent wave excitation is a good way to determine the relative distance between an excitable moiety, and a surface boundary.
Evanescence biosensor techniques are known in the art, but have not been generally used for membrane bound analytes. For example, U.S. Pat. No. 6,316,274 teaches multi-analyte biosensor methods using fluorescence moieties excited by evanescence illumination. Other representative prior art includes U.S. Pat. Nos. 6,274,872; 5,512,492; and 6,395,558.
Lipid membrane and liposome technology: As first described by Singer and Nicolson (Science 1972 Feb 18; 175(23):720-31), biological membranes are composed of a hydrophobic lipid bilayer, with membrane proteins existing as compact xe2x80x9cice bergxe2x80x9d like structures floating embedded in this lipid bilayer, which is about 6 nm thick. Due to the weak nature of hydrophobic bonds, lipids and integral membrane proteins are able to move freely (lateral mobility) within the plane of the lipid membrane. In particular, when exposed to forces from external ligands, such as bivalent antibodies, lectins, etc., membrane proteins and other lipid components are able to diffuse together to form xe2x80x9cpatchesxe2x80x9d or xe2x80x9ccapsxe2x80x9d on the membrane surface.
Because biological lipid membranes are highly complex structures with many different types of membrane proteins, biochemists and molecular biologists typically find it preferable to work with simplified synthetic lipid membranes, reconstituted from purified components. Such synthetic membranes can be synthesized by simple methods, since they tend to spontaneously form when lipid mixtures (which may also contain membrane proteins) are dissolved in detergents, and the detergent then gradually removed by dialysis or other process. The resulting synthetic membrane structure typically forms as a xe2x80x9csoap-bubble-likexe2x80x9d structure called a xe2x80x9cliposomexe2x80x9d or xe2x80x9cphospholipid vesiclexe2x80x9d. The walls of the liposome consist of lipid bilayers, and the membrane proteins typically insert themselves into the artificial lipid bilayer with the correct, or nearly correct, conformation. Depending upon the synthetic process, liposomes can be formed with diameters ranging from about 50 nm to 5,000+ nm.
Artificial liposomes (phospholipid vesicles) containing lipids and protein can be created by a number of methods, including cadmium synthesis (Thromb Haemost 1980 Aug 29; 44(1): 12-5); dialysis against octyl glucoside (Biochemistry 1986 Jul 15; 25(14): 4007-20); deoxycholate (Biochem J 1997 Jul. 17; 165(1): 89-96), and numerous other methods.
Membrane proteins: Many biologically relevant proteins are transmembrane proteins. These proteins exist embedded in membrane lipid bilayers, and typically can best be studied while associated with intact membranes. These transmembrane proteins include the 7-transmembrane proteins (reviewed by Kilpatrick et. al. J Cell Sci 2001 February; 114(Pt 4):629-41), and their medically relevant subfamily, the G-protein cell receptors (GPCR family), (reviewed by Woodside, Sci STKE 2002 Mar 19;2002(124):PE14). Other relevant transmembrane proteins include the integrin family, the cadherin family (reviewed by Angst et. al, J Cell Sci 2001 February; 114(Pt 4):629-41), and many others.
ADMET assays: The current art in in-vitro ADMET assays is described by Darvas and Dorman in High-Throughput ADMETox Estimation: In Vitro and In Silico Approaches, (2002) BioTechniques Press, Eaton Publishing, Westborough, Mass.
An ideal membrane receptor microarray would be both physiologically realistic and biochemically well defined. To do this, an ideal microarray should present membrane targets in an environment that, from both sides of the lipid bilayer, enables receptor binding proteins and other cofactors to interact in a normal manner. Additionally, an ideal membrane microarray should be able to detect the binding reactions between large numbers of different low molecular weight drug candidate ligands, and different membrane receptors. Finally, an ideal membrane receptor microarray should function using ultra-small quantities of candidate drug ligands, and ideally also be reusable.
Here, a novel type of membrane-receptor reagent, suitable for high performance membrane receptor microarrays, is taught. As will be discussed in later sections, this type of microarray may have high utility for HTS drug discovery and development. For example, a comprehensive GPCR microarray, containing a large number of different GPCR families and common GPCR variants, would detect unwanted cross-reactions at the earliest stage of the drug discovery process. This would greatly facilitate the development of highly specific and well-targeted new drugs.
The invention consists of a reagent system for monitoring the interaction between one or more membrane receptors of interest (here designated as xe2x80x9ctarget membrane receptorsxe2x80x9d), and one or more experimental ligands. These experimental ligands may be drugs, drug candidates, receptor agonists, antagonists, inhibitors, ect., and are here designated as xe2x80x9ctest ligandsxe2x80x9d This reagent system consists of:
(1) A fluid lipid membrane containing a target membrane receptor molecule labeled with a moiety that produces a detectable signal upon receiving excitation energy, and in which the fluid lipid membrane acts as a flexible tether for the receptor.
(2) A xe2x80x9creagent ligandxe2x80x9d tethered to a surface that emits energy capable of exciting the receptor""s detectible signal emitting moiety. This excitation energy diminishes sharply in intensity as distance from the surface increases. This reagent ligand binds to the target membrane receptor molecule in a reversible manner. This reagent ligand is always present in the reagent portion of the system, and it""s binding to the target membrane receptors may, or may not, be disrupted depending upon the presence and properties of the unknown xe2x80x9ctest ligandxe2x80x9d.
(3) Anchor linking means that bind the lipid membrane and the surface into a single linked structure. This structure utilizes the fluid nature of the lipid membrane to allow the target membrane receptors to associate and dissociate from their binding site on the reagent ligands. The geometry and the tethering action of the fluid lipid membrane is such that if the bond between the target membrane receptor and the reagent ligand is disrupted, the target membrane receptor is free to diffuse far enough away from the energy emitting surface so as to produce a significant change in the amount of excitation energy received by the moiety associated with the target membrane receptor.
Typically, the lipid membrane is in the form of a liposome. In use, the reagent system is typically exposed to one or more test ligands. These test ligands may either enhance or disrupt the association between the target membrane receptor and the surface bound reagent ligand. The ability of the test ligand to modulate this association is thus monitored by observing changes in the detectable signal emitted by the moiety labeled target membrane receptor.
Usually, the liposome-associated target membrane receptor molecule will be a transmembrane protein that is of biological interest, such as the 7-transmembrane proteins (particularly xe2x80x9cGPCRxe2x80x9d proteins), toll-like receptors, transport proteins, biological response modifier receptor proteins, coagulation factors, immune response receptors, enzymes, and other biologically interesting cellular receptors.
The reagent ligand that is tethered to the surface may be any molecular entity that binds to the liposome-associated target membrane receptor molecule. Usually, the target membrane receptor will be a receptor for biological signaling molecules, such as a GPCR receptor. In this case, the bound reagent ligand will often be the natural target, an agonist, or a synthetic analog of the biological signaling molecules that are bound by the target membrane receptor.
Alternative binding reactions are also possible, however. For example, the liposome bound target membrane receptor may be an enzyme. In this case, the reagent ligand may be a substrate or inhibitor of the enzyme. Alternatively, the liposome bound target membrane receptor may itself be an enzyme substrate. In this case, the reagent ligand may be an enzyme, or a molecule with similar binding properties as the natural enzyme that reacts with the target membrane receptor.
As yet another alternative, the liposome bound target membrane receptor may be an antigen, and the reagent ligand may be an antibody that binds to the antigen.
As a third alternative, the target membrane receptor may be a transport protein, such as an ABC drug transporter protein, and the reagent ligand may be a ligand or analog to a ligand that is normally transported by the transport protein.
As previously discussed, the target membrane receptor will usually be labeled with a moiety that is capable of emitting a detectable signal when exposed to an outside energy source. The lipid membrane in turn will be tethered to an energy-emitting surface, where the energy emitted by the surface changes sharply as a function of distance away from the surface. Also tethered to this surface (by different means) will be a reagent ligand that binds to the target membrane receptor. Due to the fluid nature of the lipid membrane, the resulting target membrane receptor and surface-bound reagent ligand are free to associate and dissociate. Yet both remain attached to the energy-emitting surface.
The geometry of the membrane, and the energy-emitting surface, are chosen so that the detectable signal-emitting moiety on the target membrane receptor is exposed to differing levels of excitation energy depending upon the binding or non-binding of the target membrane receptor to the surface-bound reagent ligand.
A number of different geometric configurations and detection schemes are feasible for these purposes. One particularly favored embodiment is the use of lipid membranes arranged in a spherical liposomal configuration. These spherical liposomes may be tethered to an optical surface that exposes the liposomes to evanescent wave excitation. Here, there will be a gradient in excitation energy between the surface side of the liposome, and the distal side of the liposome. The distribution of target membrane receptors on the liposome surface can thus be assessed by the relative intensity of the signal generated by the detectible signal emitting moieties that are bound to the target membrane receptors.
Other membrane configurations, e.g. lipid layers mounted on a second surface that projects away from an energy emitting surface, and/or other excitation sources (e.g. electrochemiluminescence, chemical gradients, electron transport, resonance energy transfer, etc.), where there is a sharp decrease or increase in energy transfer as a function of distance from the surface, may also be used.
This reagent and method exhibits a number of distinct advantages over the prior art. One distinct advantage is that all of the chemistry required to perform the desired analysis is present on the support as a single homogeneous unit. Thus for each test, no additional chemistry or processing steps (such as washing or centrifugation) are required. Because the reagent contains all necessary detection means, the test-ligands do not need to be artificially labeled in any way. The assay chemistry is inherently reusable, and, by simply applying a wash step between assays, may be reused for multiple assays with different test-ligands. The reagent and method uses several orders of magnitude less reagents than previous art. Since the reagents used in this type of test are typically quite expensive, the economic savings are considerable.
This support may contain a small or large number of different target membrane receptors arranged on different sections of the support. In the case where only a small number of different types of target membrane receptors are present, liposomes containing the target membrane receptors may be applied to the support by simple dipping, coating, spraying, or other means. Alternatively, large numbers of different target membrane receptors may be used, and the support may be a microarray or flow cell that has hundreds or thousands of different target membrane receptors. In this situation, the liposomes may be applied by common microarray sample spotting methods, such as slotted pens, jet printing, and the like. In other situations, the support may be the inside of a capillary tube, or the surface of an optical fiber, in which case more specialized fabrication methods may be required.
For evanescent detection methods, the support containing the energy-emitting surface will normally be made of a solid material, such as glass or plastic, that has an index of refraction that is significantly different from the liposome containing aqueous media. However use of non-solid supports is also possible. As an example, high index of refraction, water and oxygen permeable, porous polymers, such as the materials used for soft contact lenses, may also be suitable.
The liposome reagents will typically need to be anchored to the surface by means that are unaffected by the presence or absence of test-ligands. Additionally, the target membrane receptors on the liposome will themselves usually be tethered to the surface by reversible linkages to surface bound reagent ligands.
Although in some cases, it may be preferable, for each different liposome preparation, to perform this set of linkages by separate set chemical coupling reactions, this approach quickly becomes impractical for systems with a large number of different target membrane receptors. As a result, it is often preferable to employ an xe2x80x9cactivexe2x80x9d support surface with a number of the xe2x80x9cgenericxe2x80x9d components of the tethered receptor-ligand reagent (for example some of the tethering and/or binding components) pre-prepared on the support surface. With an active support, a user may create the final microarray by simply spotting the various liposome preparations onto the active surface. The spotted liposome reagents can then form the appropriate anchoring and reversible reagent ligand linkages with minimal additional effort.